Adenovirus (AV) is a DNA virus with double spiral without an
envelope and can lead to infections in humans with 52 different
serotypes gathered under seven subspecies (from A to G) (1,2). In
infants, the causative agent in 5-11% of acute respiratory infections is
AV. In addition, it also causes to pharyngoconjuntival fever, follicular
conjunctivitis, epidemic keratoconjunctivitis, myocarditis, hemorrhagic
cystitis, acute diarrhea, invagination and encephalomyelitis. It is the
most common cause of follicular conjunctivitis and keratoconjunctivitis.
Specifically, AV types 4, 8, 19 and 37 have been shown to be related to
epidemic keratoconjunctivitis (3).

Adenovirus infections are frequently observed in individuals with
immune deficiency, T or B cell dysfunction and hypogammaglobulinemia. In
newborns, the cytotoxic activity of T lymphocytes and natural killer
cells is decreased. In addition, B lymphocytes produce limited numbers
of antibodies in newborns and maternal antibodies are not sufficient in
premature infants. Therefore, newborns are more sensitive against AV
infections (1,4).

Adenovirus is transmitted by direct contact, aerosols and objects.
It is rather resistant against the external enviroment. Epidemic
keratoconjunctivitis has been shown to generally occur after examination
by an ophtalmologist (2).

In this report, the management of an epidemic of AV infection in an
NICU is described.

Material and Method

The AV epidemic which occured in the NICU between 09.14.2009 and
10.17.2009 was examined retrospectively In this report, the data of
medical records and the infection control committee were used. During
the epidemic, lower palpebral swab samples were obtained from the
infants with conjunctivitis. Stool sample was taken, if diarrhea was
present and nasopharyngeal swab sample was obtained from asymptomatic
infants. In symptomatic employees, lower palpebral swab samples were
obtained, if conjunctivitis was present and throat swab sample was
obtained, if upper respiratory infection was present. Throat swab
samples were obtained also from asymptomatic employees. Swab samples
were obtained from 15 patients (9 female, 6 male) and 25 physicians,
nurses and employees and were immediately sent to the laboratory in
viral transport medium (Universal transport medium (UTM) kit, Copan
Diagnostics, Brescia, Italy) abiding by cold chain rules. Direct
fluorescent antibody (DFA) test (sensitivity 86%, specificity 100%) and
"shell vial" cell culture method were simultaneously applied
in all samples. The diagnosis of adenoviral conjunctivitis was made with
clinical findings and/or positive DFA and cell cultures (5). Throughout
the epidemic, samples were obtained weekly from all infants. Weekly
samples were obtained until findings of conjunctivitis disappeared in
all infants and serologic tests and culture results became negative (33
days). Values were given as mean [+ or -] standard deviation. Fisher
test was used for comparison of ratios between groups and Mann Whitney U
test was used for comparison of mean values. A p value of <0.05 was
considered to be statistically significant. SPSS 13.0 statistical
program was used for analysis.

Results

10 of 15 infants who were cared in the NICU during the epidemic had
conjunctivitis and 5 had diarrhea and conjunctivitis simultaneously. In
5 of the infants who developed conjunctivits, ophtalmological
examination was performed 3 days ago for follow-up of retinopathy of
prematurity

Mean birth weight of 10 infants who developed conjunctivitis was
1090 g (600-1990 g); gestational age ranged between 26 weeks and 33
weeks (mean 28,5 [+ or -] 2 weeks) and the age at the beginning of the
epidemic ranged between 3 and 104 days (mean 60 [+ or -] 37 days). 3
infants among 10 infants with conjunctivits were male and 7 were female.
AV was demonstrated by positive DFA and cell cultures in 3 infants in
eye swabs and in one infant in the transtracheal aspiration.

Symptomatic and asymptomatic infants were compared in terms of
birth weight, gender, ventilator monitoring, ophtalmological
examination, number of ophtalmological examinations, age during the
epidemic, bronchopulmonary dysplasia (BPD) and corticosteroid therapy.
In symptomatic infants, age during the epidemic, the presence of
ophtalmological examination just before the epidemic, higher number of
ophtalmological examinations, use of corticosteroid before the epidemic
and presence of BPD was found statistically significantly higher
compared to asymptomatic infants. There was no statistically significant
difference between the two groups in terms of birth weight, gender and
presence of ventilatory care (Table 1). Two groups with conjunctivitis
in whom AV could be demonstrated and could not be demonstrated in
laboratory investigations were compared in terms of birth weight,
gender, ventilator monitoring, opthalmological examination, number of
ophtalmological examinations, age at the time of epidemic, diagnosis of
BPD and corticosteroid therapy and no statistically significant
difference was found (Table 2). When the epidemic started, there was no
subject who was receiving corticosteroid therapy.

For treatment of conjunctivitis, non-steroid antiinflammatory
collyria and antibiotic collyria were used in symptomatic subjects. AV
was not found in weekly follow-up swabs in symptomatic infants. During
the epidemic, no infant was lost.

In addition, conjunctivitis developed in the ophtalmologist and
five nurses and AV was evaluated to be positive by DFA method in one of
them. Others were found to be negative. In screening of the employees,
AV was found by DFA in nasophryngeal swabs in three individuals.

Epidemiology and management of the epidemic

At the time of the epidemic, a total of 15 infants were cared for
in the NICU in three rooms. In one of the infants, conjunctivitis was
observed three days after ophtalmological examination was performed for
retinopathy of prematurity. It was learned that the opthalmologist took
a sick report because of conjunctivits three days later.

On the 3rd day of the epidemic, an urgent meeting with the
infection control committee was held since conjunctivitis was observed
in a total of seven infants and five nurses and decisions on the
preventive measures were made. In accordance with the recommendations of
the infection control committee, eye swab samples were sent from
symptomatic infants, if conjunctivitis was present and stool samples
were sent, if diarrhea was present. Only nasopharyngeal swab samples
were sent from asymptomatic infants and employees. In addition, in
symptomatic employees, eye swab samples were sent, if conjunctivitis was
present and nasopharyngeal swab samples were sent, if upper respiratory
infection was present. DFA test and "shell vial" cell culture
were applied in all samples simultaneously.

In accordance with the infection control committee, contact and
droplet isolation precautions were taken in addition to known
precautions during the AV epidemic and information tables related to
these precautions were placed in the enterance of the unit and in
different parts of the service (Table 3).

For adenovirus isolation cleaning and disinfection of incubators
were defined in writing, described to the personnel and controlled
(Table 4).

Symptomatic infants were placed in the same room and their nurses
were separated. Patients who could be discharged were rapidly
discharged. Five nurses who had conjunctivitis were not allowed to work.
No more patients were admitted to the unit. The patients were taken to a
transient intensive care service in order to perform general hygienic
cleaning and disinfection of the service and airconditioning system of
the whole service was reviewed, cleaning was performed and the walls of
the service were painted again.

On the 6th day of the epidemic, cojunctivitis developed in three
more infants in the room where asymptomatic infants were placed and they
were taken to the room of symptomatic infants. These infants had not
been screened for retinopathy. Contact and isolation precautions were
taken for all symptomatic and asymptomatic infants. After the 6th day of
the epidemic no new case of conjunctivitis was observed. All infants
were checked with weekly swab samples for 33 days. Since adenoviral
diagnositic tests became negative in all infants and the signs of
conjunctivitis disappeared in all infants, the infants were transferred
from the transient intensive care service to the NICU on the 33rd day of
the epidemic. Infection control precautions were ended 27 days after the
last case was observed (on the 33rd day of the epidemic) (Figure 1).

Since the virus was thought to have entered into the unit during
opthalmological examination, a meeting with the opthalmologists was held
and decisions on precautions which should be taken during the evaluation
of retinopathy of prematurity were made in accordance with the decisions
of the American Ophtalmology Academy (Table 5).

[FIGURE 1 OMITTED]

On the 0th day, ophtalmological examination was performed in 5
infants. On the 3rd day, conjuntivitis developed in five infants who
were undergone opthalmological examination and in 2 infants who were not
undergone opthalmological exmination. On the 3rd day, contact and
droplet isolation precautions were taken. Symptomatic and asymptomatic
infants were taken to separate rooms and their nurses were isolated. On
the 6th day, conjunctivitis developed in 3 infants in the room where
asymptomatic infants were cared for and these infants were transferred
to the room where symptomatic infants were cared for. No new patient was
hospitalized in the service and no vizitors were allowed. Infants who
had no urgent problems any more were discharged and families were
informed about precautions. Conjunctivitis developed in the father of
one of the symptomatic infants after discharge. On the 10th day,
patients were transferred to a transient intensive care unit. The whole
service was disinfected. No other cases were found. Swab samples
obtained were found to be negative. On the 33rd day of the epidemic,
patients were transferred back to the NICU. Contact and droplet
isolation precautions were ended, since all infants were asymptomatic
and AV could not be demonstrated by weekly DFA and cell culture.

Discussion

Adenoviruses have long been associated with nasocomial infections
and many epidemics have been reported especially in intensive care
units. Epidemics have commonly been reported in NICUs. The mortality
rate of the epidemics were demonstrated to be lower with AV type 8 and
higher with type 30 and type 7 due to respiratory failure caused by
pneumoniae (4,6,7). Therefore, it has been thought that the severity of
the epidemics may be related to the serotype of the virus. No infant was
lost in the epidemic in our unit. Adenovirus typing could not be done,
since there is no center which can perform this investigation in our
country. Samples were kept for later evaluation. Since no infant was
lost during the epidemic in our unit, pneumonia was not observed and the
most common agent of viral conjunctivitis was found to be AV type 8 in a
study performed in our country, it was thought that the agent might be
AV type 8 (8).

In some studies, it was suggested that corticosteroids might cause
the clinical state to detoriorate in premature infants infected with AV
or even lead to mortality (7). Since conjunctivitis was the finding at
the time of the epidemic, no other risk factor was found except for
ophtalmological examination. When symptomatic and asymtomatic patients
were compared in terms of the presence of BPD before the epidemic and
use of corticosteroid because of BDP, BDP and use of corticosteroid were
significantly found to be more common in infants with conjunctivitis.
However, no patient was using corticosteroid during the epidemic. We
thought that this prevented severe symptoms and mortality in these
infants.

Since retinopathy examination was performed at 4-6 months of age
and every 1-2 weeks until the retina matured in symptomatic infants,
their ages were statistically significantly older compared to
asymptomatic infants and their number of retinopathy examinations was
higher.

Various therapies have been tried during adenovirus epidemics and
different results have been found. It has been thought that intravenous
immunglobulin (IVIG) might prevent viral infection, since it includes
antiviral antibodies. However, in a contrary study, IVIG used to prevent
viral infections was shown to cause AV epidemics (9). In addition,
commercial IVIGs are not expected to include sufficient antibodies
against rarely seen AV serotypes. The benefit of ribavirin or recently
sidovir has also been demonstrated (10). Since no systemic symptoms were
observed in our patients during the epidemics, IVIG, ribavirin or
sidovir treatment was not administered. Only non-steroid
antiinflammatory collyria and antibiotic collyria were used for
treatment of conjunctivitis.

As far as the literature reveals, in three of AV epidemics observed
in NICUs, the AV epidemic was demonstrated to have occured after
opthalmological examination. The causative agent was commonly reported
to be adenovirus type 8 (4,7,11).

It was thought that our patients were contaminated by adenovirus
during opthalmological examinations and became infected, because
conjunctivits was observed in the patients 3 days after ophtalomological
examination and the opthalmologist also developed conjunctivitis
simultaneously. When infants with and without conjunctivits were
compared, the number of opthalmological examinations was found to be
statistically significantly higher in infants with conjunctivitis. When
retrospective evaluation was made, it was found that masks and gloves
were not used during ophtalmological examinations at that time and
instruments were found to be insufficient and not disinfected. It was
thought that these defects were involved in spreading of the epidemic.

Adenovirus keeps its activity on plastic and metal surfaces for 49
days and on clothes and paper for 10 days and it is excreted in stool
for long-term (2,11). AV was not found in stools examined during the
epidemic and later. When the epidemic was started, admission of patients
to the unit was stopped (for 33 days) until AV was found to be negative
in DFA test and cell culture. Early recognition of the epidemic,
performing necessary isolation precautions meticulously, stopping of
admission of patients to the unit, transferring the unit and detailed
cleaning prevented the spread of the epidemic and lengthening its
duration.

Meetings were held with ophtalmologists and opthalmological
examinations which will be performed in the unit were provided to be
performed in accordance with written standards. As a result of all
precautions a second epidemic did not occur and no patient was lost.

Adenovirus epidemics require the services in NICUs to be closed and
very serious precautions to be taken and the risk of recurrence is high.
Continuous education is the main component for protection from
infections.

Table 1. Comparison of the characteristics of 10 infants who were
symptomatic (conjunctivitis-gastroenteritis) and 5 infants who were
asymptomatic during the epidemic
Characteristics Symptomatic
Birth weight (g) 1090 ([+ or -] 426)
Gender (male) 3(30%)
Gestational age (weeks) 28.3([+ or -] 2.3)
Ventilator treatment at 7 (70%)
the time of infection
Age at the time of infection (gun) 58.2 ([+ or -] 37)
Number of opthalmological examinations 2.8 ([+ or -] 2,4)
Bronchopulmonary displasia (%) 8 (80%)
Use of corticosteroid (%) 8 (80%)
Opthalmological examination 7 (70%)
Characteristics Asymptomatic p
Birth weight (g) 1248 ([+ or -] 355) 0.32
Gender (male) 3 (60%) 0.32
Gestational age (weeks) 29 ([+ or -] 1.8) 0.53
Ventilator treatment at 2 (40%) 0.32
the time of infection
Age at the time of infection (gun) 11 ([+ or -] 11) 0.014
Number of opthalmological examinations 0 0.020
Bronchopulmonary displasia (%) 0 0.007
Use of corticosteroid (%) 0 0.007
Opthalmological examination 0 0.026
Table 2. Comparison of four infants with conjunctivitis in whom AV
could be demonstrated and in 6 infants with conjunctivitis in whom
AV could not be demonstrated
Characteristics Serology (+)
Birth weight (gr) 1030 ([+ or -] 476)
Gender (male) 1(25%)
Gestational age (weeks) 28.5 ([+ or -] 2.4)
Ventilatory treatment at 4 (100,)
the time of infect on
Age at the time of infection (days) 83 ([+ or -] 16)
Number of opthalmological examinations 3.7 ([+ or -] 1)
Bronchopulmonary dysplasia (%) 4 (100%)
Use of corticosteroid (%) 4 (100%)
Ophtalmological examination 4 (100%)
Characteristics Serology (-) p
Birth weight (gr) 1130 ([+ or -] 433) 0.454
Gender (male) 2 (33%) 1
Gestational age (weeks) 28.5 ([+ or -] 2.3) 0.91
Ventilatory treatment at 3 (50%) 0.20
the time of infect on
Age at the time of infection (days) 45 ([+ or -] 39) 0.20
Number of opthalmological examinations 1.7 ([+ or -] 2.4) 0.12
Bronchopulmonary dysplasia (%) 4 (66%) 0.46
Use of corticosteroid (%) 4 (66%) 0.47
Ophtalmological examination 3 (50%) 0.20
Table 3. Droplet and contact isolation precautions
1. Hands should be definetely washed and/or hygiene should be
provided by alcohol-based hand disinfectant before contact with the
patient.
2. To prevent cross-infection hands should be definetely washed and
hygiene should be provided by alcohol-based hand disinfectant
before passing form one patient to another.
3. Before contact with the patient gloves, mask, protective gown
and protective eyeglasses should be worn (Picture 1). Protective
eyeglasses should be disinfected with 70% alcohol after use.
4. Before passing to different parts during body care, hands should
be washed and gloves should be changed.
5. Laundry washed in the unit should be washed at at least
71[degrees]C and for 25 minutes. Clean and dirty laundry should be
kept in different places.
6. Laundry which will go to the laundry room should be transferred
in a red bag.
Table 4. Cleaning and disinfection of incubators for isolation of
adenovirus
1. The inside and outside of incubators should be cleaned with
acid-based disinfectant daily.
2. Cleaning should be done twice a day and additionally whenever
contamination is observed.
3. Separate cleaning cloths should be used for each incubator.
4. Cleaning should be directed from the lower part to the superior
part in incubators.
5. Outer lids and cushions and arms surrounding these lids which
are the most common contamination regions should be wiped with a
disinfectant (preferably 70% alcohol) twice a day.
6. If the surfaces of the beds have lost integrity, they should be
changed with new beds. Damage to the matresses of the patient beds
prevents efficient disinfection and sterilization.
7. After use and after each infant exchange cleaning and
disinfection of incubators and open beds should be performed.
8. If the infants are being monitored for long-term their
incubators are disinfected at least once a week (for infants lower
than 1000 g every five days). Firstly all separable parts of the
incubator should be removed, washed by brushing and cleaned by
rubbing with detergent. All parts of the incubator should be
disinfected with 5000 ppm chlorine solution.
9. Incubators should be ventilated before reusing. If the
incubators will not be used, complete drying should be provided by
heating for 24 hours after disinfection without putting water in
the moisturizer part.
10. Containers into which water is placed should be sterilized once
a week or before each infant exchange and filled with distilled
water or sterile water every 24 hours.
11. If a fan is present, it should be cleaned and disinfected.
Table 5. Rules which ophtalmologists should abide by in the
neonatal intensive care unit
1. Ophtalmologists should wear gowns and masks before entering the
unit.
2. During ophtalmological examination glowes should be used and
changed for each patient.
3. Instruments used should be kept in 70% alcohol liquid for 10
minutes and this liquid should be changed twice a day.
4. A separate blepharostate should be used for each patient.

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